Project Summary/Abstract My laboratory studies gene regulation dynamics in mouse embryonic stem cells and fibroblast tissues. We use these model systems to investigate the mechanics of chromatin regulatory pathways that provide stability to gene expression profiles through cell division allowing for mammalian tissue identity. Preservation of heterochromatin-mediated gene repression is critical for development and it is dysregulated in a number of human diseases. Yet, the molecular mechanics of formation and memory of heterochromatin repression domains are poorly understood. To understand how these regulatory processes function in vivo, we developed a novel platform that allows individual chromatin modifying activities such as Heterochromatin Protein-1 (HP1) to be recruited with high temporal control to native chromatin substrates. We have recently improved this system to allow us to rapidly change the structure of the endogenous chromatin substrate in order to explore the regulation of a diverse range of promoter and gene structures. The long-term goal of this project is to understand the mechanism of HP1-mediated gene repression and to determine the key features that provide heterochromatin stability through successive cell generations. Specifically, in this work we examine: (1) the influence on heterochromatin assembly of chromatin structural features such as: promoter transcriptional activity, DNA methylation, and histone posttranslational modifications, (2) the influence of these same chromatin features on the durability of heterochromatin gene repression. Additionally, using high throughput screening we have discovered novel inhibitors that disrupt HP1 mediated gene repression. We will develop these small molecule probes and use these compounds to define the role of individual enzymatic activities in heterochromatin assembly and durable gene repression. At the conclusion of these studies, using a combination of chemical approaches and novel in vivo tools, we will provide a new generalizable model for how HP1-mediated heterochromatin is assembled and maintained in living cells.